Project Summary Indirect airway hyperresponsiveness (AHR) is a fundamental feature of asthma, yet the immunopathology of this critical aspect of asthma remains incompletely understood. In contrast to other features of asthma such as direct AHR to methacholine challenge and airflow obstruction that are present in other lung diseases, indirect AHR is a unique and specific feature of asthma. We have examined the pathogenesis of exercise-induced bronchoconstriction (EIB) as a prototypical feature of indirect AHR. In cross sectional studies, EIB is present in 30-50% of subjects with asthma, and can be ascertained precisely by a dry air exercise challenge test. In addition, subjects with EIB in the absence of other features of asthma have a high risk of progression to other aspects of the asthma syndrome, further verifying the fundamental relationship between indirect AHR and asthma. Our research on the pathogenesis of EIB has revealed that subjects with EIB have high levels of eicosanoids such as leukotrienes (LT) in their airways, as well as epithelial shedding into the airway lumen. Following exercise challenge, we have identified mast cell (MC) degranulation and sustained release of eicosanoids. As we have worked to unravel the basis of these alterations in the airway function, we have identified a secreted phospholipase A2 (sPLA2) that serves as a regulator of eicosanoid synthesis and is elevated in the airways of subjects with asthma, and discovered infiltration of the airway epithelium with MCs that have a distinct phenotype in subjects with EIB. Thus, the overall goal my research program is to understand the alterations in the airways that lead to indirect AHR in humans. Based on our initial observations, we hypothesize that epithelial-derived sPLA2 group X (sPLA2-X) serves as a key regulator of innate cells including MCs, leading to the generation of products such as LTs that mediate bronchoconstriction through the sensory nerves, leading to goblet cell degranulation and smooth muscle constriction. In the first aim, we use airway samples and primary epithelial cell culture models to examine the regulation and function of epithelial sPLA2-X. In the second aim, we determine differences in the number and proliferation potential of MC progenitors in the airways, and whether these cells serve as a critical source of IL-13 in asthma. In the final aim, we use design-based stereology to examine the cellular and structural basis of indirect AHR in asthma using airway biopsy samples from informative groups of subjects with and without asthma. These projects will move the field forward through a better understanding of the structural and molecular basis for indirect AHR in asthma, and will serve to train the next generation of patient-oriented researchers in this important area.